2. 2 | WHAT IS DRUG RESISTANCE?
There are two general classes of resistance, intrinsic and acquired.
Intrinsic resistance occurs due to overt pre-existing factors, including
variations in protein expression levels (such as increased expression of
the P-gp [MDR1] transporter), epigenetic modifications (including by
the long non-coding RNA (lncRNA) HAND2-AS1 and chromatin modi-
fier Jarid1A (lysine demethylase 5A)) and somatic mutations (such as
in Ras) (Burrell et al., 2013; Gruber et al., 2012; Marusyk et al., 2012;
Sharma et al., 2010). Changes conferring drug resistance often co-
exist, resulting in a resistance heterogeneity similar to that seen in the
original disease itself (Gerlinger et al., 2012; Ramirez et al., 2016).
Acquired resistance is thought to occur for a number of reasons,
including through pre-existing (but initially undetectable) and de novo
mutations (Bhaduri et al., 2020; Russo et al., 2019). This is often
identified weeks to months after treatment has commenced (Santoni-
Rugiu et al., 2019). In recent times, the concept of disease clonal
heterogeneity has provided greater insight into the causes of acquired
resistance, suggesting that chemoresistance and disease relapse occur
as a result of minor subclonal populations. Given that these likely
contribute to the heterogeneous nature of cancer, it seems likely that
these also contribute to disease re-emergence and relapse (Bonnet &
Dick, 1997; Gerlinger et al., 2012; Pattabiraman & Weinberg, 2014;
Roy & Cowden Dahl, 2018; Seth et al., 2019). Within these minor
clonal populations, mutations conferring drug resistance may exist at
the early stages of disease but remain dormant and undetectable upon
first presentation (Pietrantonio et al., 2017; Russo et al., 2018). When
the bulk of the cancer is eliminated as a result of initial chemotherapy
targeted at overt mutations, cells from this minor subclone proliferate
and become dominant in the tumour bulk (Jones et al., 2019;
McMahon et al., 2019).
A second, related, resistance mechanism involves a very rare
subpopulation of cells, known as cancer stem cells. Cancer stem cells
were first described in acute myeloid leukaemia but have since been
applied to many other cancers including (but not limited to) breast
cancer, colorectal cancer, myeloma and pancreatic adenocarcinoma
(Bonnet & Dick, 1997; Koury et al., 2017; Lapidot et al., 1994).
Cancer stem cells have unique properties compared to bulk tumour
cells. They are undifferentiated and have strong self-renewal and
proliferative capabilities and typically remain in a quiescent state,
thus avoiding chemotherapy targeted at rapidly dividing cells (as in
bulk tumour cells). However, they do have the proliferative capacity
to maintain and expand the tumour burden, as bulk tumour cells are
eliminated (Jordan et al., 2006). These “stemness characteristics”
render this subset of cells intrinsically resistant to chemotherapy,
with distinct immunophenotypic and molecular signatures (Bonnet &
Dick, 1997). This includes increased expression of efflux trans-
porter P-gp (MDR1) and the ability to repair damaged DNA, a
common method of inducing cancer cell death (Dean et al., 2005;
Pattabiraman & Weinberg, 2014).
Certain pathways up-regulated in cancer stem cells have been
linked to the increased self-renewal capacity seen in this subset of
cells. Key examples include the Wnt/β-catenin and Hedgehog
(Hh) pathways, as well as the Ras-dependent MAPK and PI3K/AKT
pathways. Of these, the Wnt/β-catenin pathway is perhaps the most
associated with cancer stem cells. Briefly, at a physiological level, Wnt
signalling is not active and β-catenin is ubiquitinated and sent for
proteasomal degradation following interaction with GSK3β, APC and
Axin. In cancer stem cells however, activation of Wnt signalling
inhibits formation of the β-catenin–GSK3β–APC–Axin complex,
stabilising β-catenin. This translocates to the nucleus whereby it
stimulates transcription of proliferative genes, including c-Myc. This
promotes proliferative signalling, increasing the self-renewal capacity
of cancer stem cells (Krausova & Korinek, 2014; Moon et al., 2014).
Although still ambiguous, it has been reported that β-catenin
stabilisation can promote Ras stabilisation and protects Ras against
proteasomal degradation (Jeong et al., 2018; Lee et al., 2018). This in
turn promotes MAPK and PI3K pathway signalling, further contribut-
ing to the self-renewal capacity of cancer stem cells.
Alternatively, up-regulation of the hedgehog (Hh) signalling
pathway has been shown in cancer stem cells. Whilst there are many
facets to this pathway, its activation can stimulate up-regulation of
stemness-associated transcription factors including NANOG, OCT4
and SOX2, thus promoting self-renewal (Boyer et al., 2005; Po
et al., 2010; Takahashi & Yamanaka, 2006; Zhu et al., 2019). Increased
activation and stabilisation of these genes (through either Hh signal-
ling or biochemical stresses) have been implicated in the dedifferentia-
tion of bulk tumour cells to cancer stem cells (Herreros-Villanueva
et al., 2013; Kumar et al., 2012).
Interest in epigenetic remodelling has grown considerably in the
last decade and it has considerable effects in promoting drug resis-
tance. lncRNAs are also implicated in survival of cancer stem cells. In
hepatocellular carcinoma, the lncRNA HAND2-AS1 is up-regulated
and stimulates the self-renewal capacity of cancer stem cells, through
activation of the bone morphogenetic protein (BMP) signalling path-
way. BMP activation has in turn been shown to induce
chemoresistance in other cancer models, such as lung cancer (Gruber
et al., 2012; Wang et al., 2015). Indeed, this pathway is also regulated
by the fusion gene CBFA2T3-GLIS2 and the Hh and JAK-STAT signal-
ling pathways (Gruber et al., 2012; Okada et al., 2014).
Taken together, it is evident that many signalling pathways and
associated factors play a critical role in mediating drug resistance.
Identifying commonalities between these pathways could present an
opportunity to reduce the incidence and impact of drug resistance.
Although the incidence of Ras mutations in cancer stem cells is rela-
tively low, Ras-mediated pathways have been implicated (Corces-
Zimmerman et al., 2014). In addition to the aforementioned β-catenin-
mediated stabilisation of Ras, stabilised OCT4 and SOX2 expression
has also been shown in response to AKT activation, by extracellular
biochemical and radiation stresses (Maiuthed et al., 2018; Park
et al., 2021). Indeed, activation of the MAPK pathway through the
Ras–Raf interaction promotes increased expression of SOX2 and
NANOG (Chan et al., 2018; Du et al., 2019). Furthermore, correlation
has been seen between up-regulation of the MAPK, PI3K and BMP
pathways in drug-resistant lung cancer (Wang et al., 2015). Thus, not
only can these pathways contribute to an increased cellular
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3. proliferation rate in bulk cancer, but they can also assist with the self-
renewal and stemness properties evident in cancer stem cells. Perhaps
unsurprisingly, inhibition of the Hh signalling pathway, when com-
bined with inhibition of mTOR (downstream of AKT), has been seen
to eliminate cancer stem cells in pancreatic cancer (Mueller
et al., 2009; Prieur et al., 2017). Furthermore, MAPK kinase (MEK)
inhibition has been implicated in reduced pancreatic cancer stem cell
survival (Walter et al., 2019).
3 | KEY EXAMPLES OF DRUG RESISTANCE
IN CANCER
In recent years, resistance has been documented amongst many can-
cer therapeutics, including traditional chemotherapeutic agents, more
novel small molecule inhibitors and monoclonal antibodies (Caiola
et al., 2015; McMahon et al., 2019; Pietrantonio et al., 2017). The
origin of this resistance (intrinsic or acquired) varies considerably
between drugs and so advances have been made in treatment stratifi-
cation, based on a patient's mutational status, for some therapies. For
example, vemurafenib, the BRAF inhibitor, is restricted to melanoma
patients with the BRAF V600E mutation (Hopkins et al., 2019). Like-
wise, the monoclonal antibody panitumumab is only recommended
for patients with wild-type Ras (Amado et al., 2008). However, as
mentioned previously, some mutations are only detectable when
patients relapse, such as the emergence of fms-like TK 3 (FLT3) or
NRAS mutations in acute myeloid leukaemia patients (Man
et al., 2012; McMahon et al., 2019; Piloto et al., 2007; Smith
et al., 2017). Many of these mutations occur downstream of the site
targeted by the drug. For example, since Ras operates downstream of
the EGF receptor (EGFR), Ras mutations play a role in rendering
cetuximab and panitumumab (which bind and inhibit EGFR) ineffec-
tive as the constitutive activation is not inhibited by the drug
(De Roock et al., 2010; Li et al., 2015; Zhao et al., 2017). However,
some of these putative resistance-causing mutations can only be
detected at relapse, once they have expanded from only existing in a
minor, undetectable subclone (as they did at diagnosis). Thus, it is
difficult to predict which patients will develop these resistance
mutations, meaning initial treatment stratification is difficult. Thus,
combination therapy between these established agents and novel
agents targeting these common mutational sites may be a way
forward in preventing resistance before it occurs, an example of which
is the combination of BRAF and MEK inhibitors, vemurafenib and
cobimetinib (Hopkins et al., 2019).
4 | INTRODUCTION TO Ras
As eluded, Ras mutations underpin resistance to a variety of therapies.
KRAS is most commonly mutated of the three Ras isoforms and is
particularly frequently mutated in lung and pancreatic cancer (Moore,
Rosenberg, et al., 2020; Prior et al., 2020). However, patterns of Ras
mutation differ between cancers and NRAS is the most frequently
mutated RAS gene in acute myeloid leukaemia. Also, HRAS accounts
for most Ras mutations in head and neck squamous cell carcinoma
(Prior et al., 2020). Indeed, this variation carries varying prognoses of
Ras-mutated cancer, with KRAS mutations conferring the lowest over-
all survival 5 years post diagnosis (43%) and HRAS mutations confer-
ring the highest (63%) (Figure 1) (Cerami et al., 2012; Gao
et al., 2013).
Most oncogenic mutations in Ras occur in the residues G12, G13
and Q61, which are located in a region conserved between HRAS,
KRAS and NRAS (Figure 2a). They occur within the effector lobe,
which comprises the first 85 amino acids and is a region of complete
sequence identity between the different RAS isoforms. Most key
interaction sites occur within the effector lobe, including nucleotide
and effector interaction sites, as well as the switch regions that medi-
ate effector interactions (Figure 2b). There is 90% similarity in the
next 80 amino acids (allosteric lobe). The only considerable sequence
differences occur at the C terminal end of the protein, known as the
hypervariable region. This is the region in which post-translational
modifications occur and membrane-targeting sequences are found
(Hobbs et al., 2016). Initial attempts at direct pharmacological
targeting of Ras centred around inhibiting these post-translational
modifications, including farnesylation (Cox & Der, 2002; Whyte
et al., 1997). However, more recent attempts have considered the
effector lobe and structures within this to be better therapeutic
targets, as discussed later (Canon et al., 2019; Janes et al., 2018;
Ostrem et al., 2013).
Ras is activated or inactivated when bound to GTP or GDP,
respectively. Oncogenic mutations increase the frequency of GTP-
bound (active) Ras, through two key mechanisms. This is either
through decreasing the affinity of Ras for GTPase-activating proteins
(GAPs), which stimulate the intrinsic GTPase activity of Ras and there-
fore facilitate hydrolysis of GTP to GDP (off/inactivating mechanism)
or by decreasing the need for guanine nucleotide exchange factors
(GEFs), which mediate the on/activating mechanism (Smith
et al., 2013; Wittinghofer & Waldmann, 2000). These differences
depend on multiple factors including the specific amino acid mutation
and resulting protein conformation (Hunter et al., 2015; Miller &
Miller, 2012; Poulin et al., 2019; Prior et al., 2012; Smith et al., 2013).
Some mutations (including G12C) promote rapid cycling between
the inactive and active states, an area which has been exploited by a
range of novel Ras-targeting drugs (Fell et al., 2020; Hunter
et al., 2015; Patricelli et al., 2016).
5 | Ras SIGNALLING—REGULAR AND
ONCOGENIC
Ras is involved in key pathways regulating cell proliferation,
differentiation and sensitivity to apoptosis, perhaps the most notable
being the MAPK pathway and the PI3K/AKT pathway. KRAS has also
been implicated (albeit in a more indirect way) in a range of other
pathways, including the Wnt/β-catenin pathway and the nuclear
factor erythroid 2-related factor 2 (NRF2) pathway (DeNicola
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4. et al., 2011; Ferino et al., 2020; Moon et al., 2014; Park et al., 2019;
Tao et al., 2014). These pathways are detailed briefly below and are
summarised in Figure 3.
Ligand binding to receptor tyrosine kininases (RTKs), such as
EGFR or FLT3, causes autophosphorylation of key tyrosine residues
on the RTK, after which adaptor proteins such as GRB2 bind to these
phosphorylated tyrosines via SH2 domains. Son-of-sevenless (SOS), a
Ras-guanine nucleotide exchange factor, then associates with GRB2
via two SH3 domains and ultimately facilitates the exchange of GDP
for GTP on RAS, thereby activating it (Freeman, 2000).
Following this, Ras stimulates recruitment of its serine–threonine
kinase effector, RAF, to the membrane, which binds through its Ras
binding domain and then proceeds to phosphorylate downstream
kinases including MEK and ERK (Marais et al., 1995; Molina &
Adjei, 2006). ERK then translocates from the cytoplasm to the
nucleus, causing the activation of many transcription factors, which
FIGURE 1 The impact of Ras alterations on disease. Data collected from 32 curated, non-redundant studies, comprising 10,967 patient
samples, as collated by the TCGA Pan-Cancer Atlas. (a) Ten-year profession-free survival (PFS) analysis for patients with and without Ras
alterations. Ras wild-type (blue) versus altered (red) overall survival. Ras wild-type median PFS = 65.88 months, Ras-altered median
PFS = 44.19 months. P < .01. (b) Ten-year PFS stratified by altered Ras isoform. KRAS (orange) median PFS = 36.72 months, NRAS (green)
median PFS = 45.67 months, HRAS (purple) median PFS = 74.89 months. P < .01. (c) Cancer types where KRAS is altered in at least 10% of cases.
(d) Cancer types where NRAS is altered in at least 10% of cases. (e) Cancer types where HRAS is altered in at least 10% of cases. Data obtained
from cBioPortal, all TCGA Pan-Cancer Atlas Studies
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7. go on to regulate a host of processes, including proliferation and dif-
ferentiation (Guo et al., 2020; Zhang & Liu, 2002).
Alternatively, activated Ras can promote the PI3K–AKT pathway.
This pathway also plays a key role in controlling survival, division and
metabolism and can be activated in many different ways (Castellano &
Downward, 2011; Mendoza et al., 2011). Ras binds and activates the
p110α catalytic subunit of PI3K, which in turn promotes transforma-
tion of the plasma membrane-bound lipid PIP2 to PIP3 and activation
of AKT by binding through its pleckstrin homology (PH) domain (Can-
tley, 2002; Castellano & Downward, 2011). AKT interacts with many
downstream effectors, including MDM2, BAX BAD and NF-κB. These
interactions regulate apoptosis (Cantley, 2002; Castellano & Down-
ward, 2011; Chang et al., 2003; Duronio, 2008). Furthermore, activa-
tion of FOXO by AKT promotes cellular metabolism (Engelman
et al., 2006). Given that the MAPK and PI3K pathways are both
strongly involved in controlling cell survival and death, it is clear that
aberrant signalling within these pathways will lead to cancer, of which
key hallmarks include sustaining proliferative signalling, enabling repli-
cative immortality and resisting cell death (Hanahan &
Weinberg, 2011).
Aside from these two classic Ras-dependent pathways, increasing
evidence suggests the implication of normal and mutant Ras function
in alternative mechanisms, such as redox homeostasis and stem cell
survival (Mukhopadhyay et al., 2020; Wu et al., 2019). The role of Ras
in cancer stem cells is yet to be fully elucidated. However, its crosstalk
with the Wnt/β-catenin pathway in tumorigenesis may suggest its
involvement in the maintenance of cancer stem cells (Moon
et al., 2014). It is understood that, in colorectal cancer, there is over-
activation of both the Wnt/β-catenin and Ras pathways, through
mutations within APC (a tumour suppressor) and KRAS, respectively
(Jeong et al., 2018). There is a synergistic effect seen with these
mutations (D'Abaco et al., 1996; Janssen et al., 2006; Jeong
et al., 2018; Margetis et al., 2017). Mutations within APC cause loss of
function of the tumour suppressor gene, whilst KRAS mutations lead
to phosphorylation of key tyrosine residues within β-catenin, causing
its accumulation within the cytoplasm. This ultimately increases acti-
vation of downstream Wnt pathway target genes, including REG4, a
marker of cancer stem cells (Hwang et al., 2020; Janssen et al., 2006).
Up-regulation of this pathway is highly associated with the increased
survival and plasticity properties seen in cancer stem cells and has
been seen in many cancers (Al-Hajj et al., 2003; Koury et al., 2017;
Lapidot et al., 1994). Taken together, it seems plausible that these
KRAS mutations may play a role in the protection of the minor subset
of cells, which likely have a key role in relapse.
Furthermore, Ras mutations can also be implicated in ROS
generation/detoxification. ROS are considered a “double-edged
sword,” capable of both helping and hindering cancer cells (Hayes &
McMahon, 2006; Wu et al., 2019). At physiological levels of ROS,
NRF2 binds Keap1 and is ubiquitinated and degraded on a regular
basis. However, upon detection of high levels of ROS (which is carci-
nogenic), NRF2 is unable to bind to Keap1 (due to conformational
changes in Keap1) and so translocates to the nucleus, where it acts as
a transcription factor for various downstream detoxification genes.
T
A
B
L
E
1
(Continued)
Name
Drug
type
Target
Development
stage
Mode
of
inhibition
Selected
common
adverse
effects
References
RBC8
Carbonitrile-based
small
molecule
inhibitor
RAL
Preclinical
Non-ATP-competitive
inhibitor,
binds
GDP-loaded
RAL,
preventing
activation
N/A
Walsh
et
al.
(2019);
Yan
et
al.
(2014)
Alpelisib
Aminothiazole-based
small
molecule
inhibitor
PI3Kα
FDA-approved
ATP-competitive
inhibitor,
binds
selectively
to
PI3Kα
Hyperglycaemia,
rash,
diarrhoea,
nausea,
decreased
appetite
André
et
al.
(2019);
Furet
et
al.
(2013)
Uprosertib
Thiophenecarboxemide-based
small
molecule
inhibitor
AKT
Phase
II
(NCT01902173)
ATP-competitive
inhibitor,
binds
AKT
and
reduces
downstream
signalling
Nausea,
vomiting,
diarrhoea,
rash
Gungor
et
al.
(2015);
Nitulescu
et
al.
(2016)
Everolimus
Macrocyclic
lactone-based
small
molecule
inhibitor
mTOR
FDA-approved
Complexes
with
FKBP12,
which
binds
mTOR
and
inhibits
activation
Leukopenia,
hypercholesterolaemia,
hyperlipidaemia
Dunn
and
Croom
(2006)
Abbreviations:
PPI,
protein–protein
interaction;
SOS,
son-of-sevenless;
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8. FIGURE 2 2D and 3D representation of the structure of RAS. (a) Physiological binding domains. (b) Key structural domains (switch regions
and lobes), with mutational hotspots G12, G13 and Q61 indicated. Redrawn from Prior et al. (2012). 3D structures based on PDB 4DST
FIGURE 3 RAS-mediated pathways and associated inhibitors. Targets of small molecule inhibitors and monoclonal antibodies used across a
range of cancers to inhibit proliferative signalling and survival of cancer cells. Figure includes examples of compounds identified in vitro, those
which have progressed into trials and those which are approved. Further detail is provided in Table 1
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9. Hence, the DNA of the healthy cells remains undamaged by ROS
(Basak et al., 2017; Gorrini et al., 2013). However, in cancer, the NRF2
pathway can be up-regulated to constitutively degrade ROS (induced
by chemotherapeutics), thereby conferring a protective effect to the
cancer cells, resulting in chemoresistance (Basak et al., 2017; Gorrini
et al., 2013). Therefore, the balance of NRF2 activation level is crucial.
In the last decade, it has been shown that KRAS G12D mutations can
increase NRF2 transcription, through activation of the TRE (12-O-
Tetradecanoylphorbol-13-acetate (TPA) response element) via the
MAPK pathway (DeNicola et al., 2011; Mukhopadhyay et al., 2020;
Shirazi et al., 2020; Tao et al., 2014). Thus, these KRAS mutations can
render the cancer cell more capable of coping with chemotherapy-
induced ROS, thereby mediating drug resistance.
6 | THE INVOLVEMENT OF Ras IN
CHEMOTHERAPY RESISTANCE
Should mutations occur as described above, it follows that one or
many of these pathways can be perturbed, leading to increased cell
proliferation, decreased cell death and promotion of cancer stem cells,
amongst other effects. The following scenarios illustrate some of
these key resistance mechanisms, highlighting the necessity for better
Ras targeting.
Platinum-based agents, such as cisplatin, carboplatin and
oxaliplatin, are used in the treatment of a variety of cancers, including
head and neck squamous cell carcinoma, testicular cancer and
non-small cell lung cancer (de Vries et al., 2020; Silva et al., 2019;
Weykamp et al., 2020). They are DNA intercalating agents that
interfere with RNA transcription and DNA replication, through cross-
linking of DNA. This results in the formation of DNA adducts, which
in turn drive the tumour cell to apoptosis. Cisplatin also induces
mitochondrial ROS, which further increase DNA damage and thus
increase the cytotoxic properties of the drug (Marullo et al., 2013;
Srinivas et al., 2019). However, there are many resistance mechanisms
associated with cisplatin, including the involvement of oncogenic
KRAS mutations (Caiola et al., 2015; DeNicola et al., 2011; Feldman
et al., 2014; Garassino et al., 2011). KRAS mutations were shown to
induce NRF2 pathway up-regulation in non-small cell lung cancer,
thereby decreasing cisplatin-induced ROS within the tumour cell, and
ultimately leading to decreased cell death (DeNicola et al., 2011). This
was supported by further work indicating oncogenic KRAS can induce
NRF2 gene transcription via the TPE response element, resulting in
the overactivation of the anti-oxidative stress pathway, rendering the
tumour cells resistant to cisplatin-induced ROS (Tao et al., 2014).
Furthermore, KRAS mutations can lead to hyperactivation of the
PI3K–AKT pathway, which is starting to be implicated as a cisplatin
resistance mechanism. As mentioned, up-regulation of the PI3K–
AKT–mTOR pathway can have multiple effects, including inhibition of
apoptosis and increased cell proliferation (de Vries et al., 2020).
Whilst there are other reasons for cisplatin resistance, Ras pathway
mutations are heavily implicated in the key mechanisms. Thus,
pharmacologically targeting Ras would provide an opportunity to
overcome many causes of this resistance.
Up-regulation of Ras-mediated pathways as a means of
chemoresistance is by no means restricted to cisplatin resistance
and is a common mechanism of resistance to TK inhibitors (TKIs),
such as those targeting RTKs including FLT3 and EGFR (Eberlein
et al., 2015; Massarelli et al., 2007; McMahon et al., 2019;
Ortiz-Cuaran et al., 2016; Piloto et al., 2007; Van Emburgh
et al., 2016). TKIs are used in a variety of cancers, including renal cell
carcinoma (RCC), colorectal cancer, acute myeloid leukaemia and non-
small cell lung cancer, to name a few examples. The mechanism of
action of TKIs involves inhibition of phosphorylation sites within the
protein, thereby preventing it exerting kinase activity on downstream
effectors (Ciardiello & Tortora, 2008; Yamaoka et al., 2018). However,
resistance to these can occur through two predominant mechanisms,
mutations within the RTK or mutations within downstream pathways
(McMahon et al., 2019; Piloto et al., 2007; Van Emburgh et al., 2016;
Yu et al., 2013). Given that Ras occurs downstream of these recep-
tors, any mutations within Ras will render the cell resistant to the TKI.
For example, studies have shown KRAS mutations render patients
resistant to gefitinib, used to treat non-small cell lung cancer (Pao
et al., 2005; Zhao et al., 2017). In a similar way, the treatment of
colorectal cancer with anti-EGFR monoclonal antibodies cetuximab or
panitumumab is only successful in a subset of patients, with many
eventually developing resistance (Pietrantonio et al., 2017). This has
been attributed to Ras mutations and variations in the EGFR extracel-
lular domain, which reduce antibody binding efficiency, ultimately
initiating relapse (Van Emburgh et al., 2016). Although cetuximab and
panitumumab are only prescribed to Ras wild-type patients, emer-
gence of mutations from undetectable, pre-existing clones can give
rise to resistance in this way, as evidenced through analysis of circu-
lating tumour DNA (ctDNA) (Amirouchene-Angelozzi et al., 2017; Diaz
et al., 2012; Misale et al., 2012). The order in which these mutations
develop/emerge is likely important in understanding (and ultimately
targeting) the process of relapse. Ras mutations often develop earlier
than EGFR variations and typically confer poorer prognosis (Van
Emburgh et al., 2016). Therefore, combatting these Ras mutations
would not only improve prognosis of Ras-mutated patients but also
provide a second therapeutic option for those that go on to develop
extracellular domain variations.
Resistance to FLT3-TKIs is also a highly prevalent issue. It is well
documented that 20–30% of acute myeloid leukaemia patients have
an internal tandem duplication in the FLT3 RTK (FLT3-ITD) causing
increased cell proliferation and decreased apoptosis, via the MAPK,
STAT5 and PI3K pathways (Hayakawa et al., 2000; Moore, Faisal,
et al., 2020; Papaemmanuil et al., 2016; The Cancer Genome
Atlas, 2013). Therefore, many different FLT3 inhibitors are at varying
stages in development to overcome the effects of this mutation.
Examples include gilteritinib, crenolanib and midostaurin (Aikawa
et al., 2020; McMahon et al., 2019; Piloto et al., 2007; Zhang
et al., 2019). These TKIs bind to the active conformation of FLT3 and
are at varying stages of approval. Gilteritinib and midostaurin are
FDA-approved while crenolanib is in Phase II trials (Galanis
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10. et al., 2014; Levis, 2017; Levis et al., 2011; Zhang et al., 2019).
Although results for these drugs have all been promising, subsets of
patients exhibit resistance. This has, in part, been attributed to Ras
mutations, reactivating the MAPK and PI3K–AKT pathways
(McMahon et al., 2019; Zhang et al., 2019). These mutations were
detected in over 30% of patients who developed resistance to
gilteritinib, with Ras variant allele frequencies also increasing post-
drug exposure in patients who responded poorly to crenolanib. Inter-
estingly, not all resistant patients had FLT3 mutations following treat-
ment either, with different mutational signatures present instead
(McMahon et al., 2019; Zhang et al., 2019). This implies a clonal selec-
tion mechanism of resistance—a minor subpopulation at diagnosis
which became dominant following treatment and elimination of the
initial tumour burden. Taken together, it seems likely these Ras
mutations, either pre-existing or de novo, may contribute to resistance
to FLT3 inhibitors, through restoration of the original disease pheno-
type by expansion of an originally minor subclone. This is perhaps
unsurprising in acute myeloid leukaemia, which arises as a result of
clonal haematopoiesis (Desai et al., 2018).
7 | OPTIONS FOR TARGETING THE Ras
PATHWAY
With improved capability of detecting minor cancer subclones,
alongside the greater understanding of the impact of Ras mutations in
various cancers, the next considerable challenge is improved pharma-
cological targeting of Ras. However, drugging Ras has proven excep-
tionally difficult. A key drawback is the lack of available binding sites
for small molecule inhibitors. The nucleotide binding site (where GTP
or GDP binds) seems a desirable pocket to target, however the
picomolar affinity with which both GDP and GTP bind, as well as their
high intracellular concentrations, effectively outcompetes the binding
of any drug at this site (Cox et al., 2014; McCormick, 2018).
Therefore, targeting alternative proteins within Ras-regulated
pathways has been strongly investigated, with positive results seen,
for example with trametinib, the MEK inhibitor. Currently approved
for patients with BRAF V600-mutant metastatic melanoma or BRAF-
mutated (V600) non-small cell lung cancer, trametinib is well tolerated
in patients (Lugowska et al., 2015; Odogwu et al., 2018) and is also
being assessed in Ras-mutant myeloid malignancies (Borthakur
et al., 2016). This compound binds to phosphorylated MEK and
inhibits its downstream effectors (e.g. ERK), despite the presence of
constitutive Ras signalling. Subsequently, aberrant growth signalling
and apoptosis inhibition is reduced (Hofmann et al., 2012). Whilst this
has shown promising results (Borthakur et al., 2016; Lugowska
et al., 2015; Odogwu et al., 2018), this compound only inhibits the
MAPK pathway downstream of MEK, so constitutive activation of
other Ras-dependent pathways (e.g. PI3K–AKT) will still occur in the
presence of Ras mutations even when treated with this drug. In this
way, cancer can persist (Jones et al., 2019; Stinchcombe & John-
son, 2014). However, if Ras were to be targeted directly, signalling of
both of these pathways would be inhibited, leading to cell death.
As often seen with many diseases, combination of trametinib with
other therapeutics to inhibit multiple pathways together may reduce
the likelihood of continued cancer signalling and potential develop-
ment of resistance to this and other drugs (Infante & Swanton, 2014;
Planchard et al., 2016; Zhou et al., 2020). However, even with the
approved combination regimen of dabrafenib (BRAF inhibitor) with
trametinib (Lugowska et al., 2015), only the MAPK pathway is
inhibited, thereby maintaining the potential for aberrant PI3K path-
way signalling, which can in itself cause resistance to MEK inhibitors
(Jaiswal et al., 2009; Sos et al., 2009; Vitiello et al., 2019).
Alternatively, positive effects have been shown in vitro and in vivo
of co-administering AKT inhibitors with dabrafenib to inhibit two
Ras-mediated pathways (Lassen et al., 2014). However, combination
of the two classes of drugs in patients did not yield significant clinical
activity in reducing resistance seen in trametinib monotherapy, with
25% of patients exhibiting grade 3–4 toxicity (Algazi et al., 2018).
Taken together, whilst downstream pathway inhibition has proved
successful, this treatment method does have considerable disadvan-
tages and may not be a long-term solution for many patients.
Therefore, there is a distinct clinical need for novel means of treating
Ras-mutant cancers, which could include the targeting of Ras itself.
Given the difficulties with targeting Ras, inhibition of elements
upstream of Ras has been investigated. This includes inhibition of
GEFs including SOS Ras/Rac guanine nucleotide exchange factor 1 (
SOS1), to reduce the likelihood of Ras maintaining its GTP-bound
state and therefore inhibiting constitutive signalling (Evelyn
et al., 2014; Hillig et al., 2019). For example, BAY293 is a first-in-class
compound with the ability to bind directly to SOS1 and inhibit the
Ras–SOS interaction and thereby downstream signalling of the PI3K
and MAPK pathways (Hillig et al., 2019). Although the in vivo bioavail-
ability for this compound was poor, the concept of Ras–SOS inhibition
could prove useful in the future, with promising high throughput in
silico and in vitro screening results serving as a proof of concept for
inhibition of Ras via this mechanism (Evelyn et al., 2014, 2015; Hillig
et al., 2019). More recently, BI-1701963, a SOS1–pan-Ras interaction
inhibitor, has reached Phase I clinical trials, the first of its kind to do
so. Modified from the structure of BI-3406, a quinazoline-derived
compound, this novel inhibitor binds to the catalytic site of SOS1,
preventing its interaction with inactive KRAS, thus inhibiting activa-
tion (Gerlach et al., 2020; Hofmann et al., 2020).
In addition, alternative, more indirect pathways are also being
targeted as a means of inhibiting Ras, which may also be able to elimi-
nate the cancer stem cell. For example, the small molecule KYA1797K
has been shown to be effective against oncogenic Ras in colorectal
cancer and erlotinib-resistant non-small cell lung cancer (Park
et al., 2019). This compound indirectly targets Ras, through inhibition
of the Wnt/β-catenin pathway, which usually stabilises Ras (Jeong
et al., 2012; Moon et al., 2014). This pathway is also up-regulated in
cancer stem cells (Malanchi et al., 2008). KYA1797K has initiated anti-
tumour effects in KRAS-mutant cell lines and a KRAS-mutated mouse
model. KYA1797K also exhibited synergy with the current first-line
therapeutic regimen (cisplatin and pemetrexed) in non-small cell lung
cancer in vitro models (Park et al., 2019). However, KYA1797K
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11. promoted apoptosis of both KRAS wild-type and mutant cells,
questioning the specificity of the drug for cancer cells whilst sparing
healthy cells. This study found both KRAS and β-catenin were
overexpressed in tumour regions compared to non-tumour regions
(Park et al., 2019). This may explain the limited toxicity seen during
KYA1797K studies, with low doses of KYA1797K having a more sub-
stantial effect on tumour cells, compared to healthy cells. Indeed, only
limited toxicity was seen during in vivo studies (Park et al., 2019).
However, protein expression varies considerably between tissues
(with KRAS particularly highly expressed in the brain) and so targeting
based on comparative expression between cancer and non-cancer
regions in one tissue may not represent overall toxicity potential
(Newlaczyl et al., 2017). Taken together, there may be a role for
KYA1797K as a concomitant therapy in non-small cell lung cancer
(as well as other cancers such as colorectal cancer). It presents a
means of eliminating both the primary cause of the disease (if KRAS-
mutated) and also minor subclones and cancer stem cells that could
give rise to resistance (Cho et al., 2020). Nevertheless, better under-
standing of the drug's effects on other tissues with high Ras expres-
sion must be gained.
8 | OPTIONS FOR TARGETING Ras—
DIRECT Ras TARGETING
Ras post-translational modifications were targeted as a means of
preventing Ras trafficking to the membrane and therefore
inhibiting downstream signalling. This included generation of
farnesyltransferase inhibitors, including lonafarnib and tipifarnib (Van
Cutsem et al., 2004). However, the effectiveness of these was
questionable, with most patients with KRAS-mutated diseases (such as
pancreatic cancer and leukaemia patients) receiving no clinical benefit
from these farnesyltransferase inhibitors (Borthakur et al., 2006;
Burnett et al., 2012; Harousseau et al., 2009; Van Cutsem et al., 2004).
Inefficacy was largely due to the redundancy mechanism of
geranylgeranyltransferase, which sufficiently modifies KRAS in the
absence of farnesyltransferase to permit its trafficking to the membrane
(Basso et al., 2006; Whyte et al., 1997). Interest in this strategy has
recently been revived with new personalised medicine approaches now
capable of identifying patients harbouring HRAS- or NRAS-driven can-
cers that are more likely to respond (Gilardi et al., 2020; Lee et al., 2020).
In recent years however, more promising steps have been made
regarding direct inhibition of oncogenic Ras. A key feature of this has
been the discovery of novel potential binding pockets for small mole-
cule inhibitors, to inhibit GEF activity or effector binding (Cruz-Migoni
et al., 2019; Maurer et al., 2012; Ostrem et al., 2013). Fragment-based
screening identified a previously undiscovered hydrophobic pocket
located between the Switch I and II regions (termed S-IIP), which was
successfully targeted by Ostrem et al. (2013) (Figure 4a). Binding of
peptide fragments was specific to G12C-mutated KRAS since the
compounds functioned through irreversible cysteine binding in this
particular pocket (and not with other cysteines found in wild-type
KRAS). Other key residues within this pocket include, but are not
limited to, V7, V9, M72, F78, Q99 and I100. In vitro models of KRAS
G12C-mutated lung cancer treated showed decreased survival upon
treatment with these compounds, with inactive Ras (Ras-GDP) levels
considerably greater than Ras-GTP. Further analysis showed that the
conformational disruption caused by binding of these fragments
reduced interactions with both SOS and effector molecules and
pathways, including B-RAF, C-RAF and the PI3K pathway (Gentile
et al., 2017; Ostrem et al., 2013).
The binding of these fragments to Ras also reduce SOS-catalysed
nucleotide exchange, a method of Ras inhibition which had been
previously explored. Compounds acting in this way either inhibited
conversion of Ras-GDP to Ras-GTP (Patgiri et al., 2011) or increased
the amount of Ras-GTP to such a level that it inhibited ERK phosphor-
ylation, since overactivation of Ras can be cytotoxic (the Ras “sweet-
spot model”) (Li et al., 2018). Either way, these compounds were
shown to inhibit the MAPK pathway but were largely tested against
Ras wild type (in the context of inhibiting the effects of RTK muta-
tions). Thus, the aforementioned compounds identified by Ostrem
et al. were revolutionary in their specificity for targeting Ras-mutated
disease.
Discovery and characterisation of this SII-P pocket have led to
the development of revolutionary KRAS G12C-selective covalent
inhibitors, including ARS-853 and latterly AMG-510 (Figure 4b–d)
(Canon et al., 2019; Patricelli et al., 2016). ARS-853 binds irreversibly
to the inactive form of KRAS G12C, preventing exchange of GDP for
GTP and therefore activation. This in turn inhibits downstream MAPK
and PI3K–AKT pathway signalling, with KRAS–CRAF interactions sig-
nificantly reduced. Moreover, in vitro evidence showed increased apo-
ptosis and cell cycle arrest in some (but not all) models tested (Lito
et al., 2016; Patricelli et al., 2016). This compound has subsequently
been fully characterised in silico and these studies revealed a dynamic
nature of the SII-P pocket, a feature which could be utilised in further
study (Khrenova et al., 2020).
Based on this work, alternative iterations of KRAS G12C inhibi-
tors have been produced. This was required since the probability of
ARS-853 locking KRAS in its inactive state in vivo was debatable,
given a lack of understanding regarding the cycling efficiency of Ras
between its inactive and active states. It had been deduced in vitro
that the G12C mutation permits rapid cycling of KRAS between these
states, hence permitting the binding of ARS-853 (Figure 4b). How-
ever, the possibility of finding the correct therapeutic window to
translate this compound to in vivo work proved complex (Janes
et al., 2018; Lito et al., 2016; Patricelli et al., 2016). Thus, alternatives
including ARS-1620 were developed to improve in vivo capability
(Figure 4c). Modifications to the ARS-853 structure resulted in
favourable pharmacokinetic (PK) properties, permitting a greater
understanding of KRAS activation status and dependency in vivo
(Janes et al., 2018). ARS-1620 is an orally bioavailable quinazoline-
based compound with limited side effects witnessed in preclinical
animal models (Janes et al., 2018; Li et al., 2018). Optimisation from
ARS-853 by inclusion of a fluorophenol, hydrophobic binding group
permitted stronger covalent (irreversible) binding within the SII-P
pocket (more specifically, interaction with H95 in this pocket), thus
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12. FIGURE 4 Evolution of novel direct RAS-targeting agents. Chemical structure and protein structures of direct RAS-targeting agents in
complex with GDP-bound (pink) KRAS. (a) Minor modification of initial compound hit 6H05, 6H05 compound 6 (purple) bound covalently to
KRAS G12C, PDB accession no. 4LUC. (b) ARS-853 (purple) bound covalently to KRAS G12C (orange), PDB accession no. 5F2E. (c) ARS-1620
(purple) bound covalently to KRAS G12C (orange), PDB accession no. 5V9U. (d) AMG-510 (purple) bound to KRAS G12C (orange), PDB accession
no. 6OIM. (e) MRTX849 (purple) bound covalently to KRAS G12C (orange), PDB accession no. 6UT0. Molecules shown in relation to the switch
regions, largely binding in SII-P. All compounds bind covalently near to mutational hotspot
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13. improving potency (Figure 4). The effects of this compound remained
mutation specific, thereby eliminating the risk of binding to KRAS wild
type in non-tumour cells and thus reduced toxicity potential. This
compound was well tolerated in patient-derived xenograft mice,
where a reduction in tumour burden through decreased Ras-mediated
downstream signalling was evident. This was the first example of an
in vivo trial using a compound targeting the SII-P pocket (Janes
et al., 2018).
From this, AMG-510 was developed, following further
modifications to the ARS-1620 structure, including addition of more
aromatic entities (Figure 4d). This enables AMG-510 to bind within a
slightly different groove of SII-P, enhancing potency and selectivity
(Canon et al., 2019). Promising preclinical in vitro and in vivo
experiments indicated arrest of the MAPK pathway and induction of a
pro-inflammatory tumour micro-environment. This compound has
since progressed into clinical trials, the first KRAS G12C-selective
inhibitor to do so. Early data indicate that out of 29 patients evaluable
for response at the time of publication, 5 exhibited partial response,
18 had stable disease and 6 had progressive disease. As with the
previous ARS-1620 animal studies, AMG-510 was generally well
tolerated, with no dose limiting toxicities recorded (Govindan, 2019;
Romero, 2020). However, larger cohorts and longer trials are
imperative in determining the true impact of this compound. Such
compounds could help combat the KRAS-mediated resistance to
monoclonal antibodies seen in non-small cell lung cancer and colorec-
tal cancer (Lièvre & Laurent-Puig, 2009; Park et al., 2019). However,
success of this compound and indeed this targeting mechanism, is
likely restricted to KRAS G12C-mutant cancer since some key
residues for binding of this compound are unique to KRAS and not
conserved between the different isoforms. Indeed, in vitro work
completed by Ostrem et al. (2013) illustrated that transduction of lung
cancer cell lines with KRAS G12V rescued the cancerous phenotype
(resistance to cell death, increased proliferation), thereby illustrating
how this mutation renders resistance to KRAS G12C inhibitors, as
expected.
Alternative KRAS G12C inhibitors are also in development and
showing considerable promise, including MRTX849 (Fell et al., 2020;
Hallin et al., 2020a). In a similar way to AMG-510 and the ARS com-
pounds discussed above, MRTX849 covalently binds to the inactive
form of KRAS, in SII-P (Figure 4e). This induces apoptosis through
down-regulation of the MAPK pathway. Interestingly, the PI3K–AKT
pathway remained relatively unaffected by MRTX849 (Hallin
et al., 2020b). In vivo trials with MRTX849 exhibited favourable phar-
macokinetic/pharmacodynamic properties (Fell et al., 2020) and both
cell line- and patient-derived xenograft modelling of pancreatic and
lung cancers also indicated up to a 30% reduction in tumour burden
(Fell et al., 2020; Hallin et al., 2020b). Individual patient case studies
from Phase I trials have also shown MRTX849 to be effective in
reducing tumour burden in both lung cancer and colorectal cancer,
although these data are largely incomplete (Hallin et al., 2020b). Taken
together, it is clear that KRAS G12C inhibitors have promise as a
means of abrogating Ras-mediated resistance, although there remain
drawbacks which need assessing, most notably, the lack of efficacy
against other Ras mutations, which are prevalent across Ras-mutated
disease.
Clearer understanding of the structure of Ras has permitted not
only elucidation of the SII-P pocket but also alternative binding sites,
including a pocket between the Switch I and II regions of Ras (termed
pocket I). Key residues available for interaction with small molecule
compounds include K5, L6, V7, V8, S39, D54, I55, L56, Y71, T74, G75
and E76 (Maurer et al., 2012; Quevedo et al., 2018). Antibody-frag-
ment-directed site exploration can be used to explore and exploit pre-
viously unconsidered drug interaction sites. In the case of Ras, this
mechanism has been used to analyse potential compound binding
sites within the previously identified pocket I that could be targeted
using small molecule inhibitors to interrupt effector proteins (such as
c-RAF and p110α) from binding to Ras via the Ras binding domain
(Maurer et al., 2012; Quevedo et al., 2018). This would provide an
alternative mechanism of abrogating the effects of oncogenic Ras
activation to those discussed previously and early results showed
effectiveness of antibody-derived compounds against a range of Ras
mutations and isoforms (Quevedo et al., 2018). However, given high
affinity binding of certain effectors to Ras (such as PI3K and B-RAF,
with 3.2- and 0.04-μM affinity, respectively) (Erijman &
Shifman, 2016), the high EC50s of the compounds identified in these
in vitro assays mean that many further modifications would be
required to convert these putative compounds into usable therapeu-
tics. Nevertheless, such antibody-derived fragments have the poten-
tial to be fused with small molecule protein–protein interaction
inhibitors to improve efficacy (Cruz-Migoni et al., 2019). Whilst many
Ras–effector interaction inhibitors have been trialled preclinically,
none have been implemented in the clinic in the context of Ras-
mutant cancer, owing to lack of efficacy or toxicity potential (Canon
et al., 2019; Keeton et al., 2017). However, crystal structure
determination showed that fusion of compounds developed through
antibody-derived fragment screening with known small molecule
protein–protein interaction inhibitors results in better binding within
pocket I, thus inhibiting Ras–effector interactions with a lower EC50.
Nevertheless, the therapeutic use of pocket I may be restricted since
such a pocket has also been detected in wild-type Ras (Cruz-Migoni
et al., 2019), thus increasing the risk for on-target toxicity.
In recent times, inhibition of the Ras–effector interaction has
been seen through competitive binding of rigosertib at the Ras bind-
ing domain. This compound elicits effects against MAPK, PI3K and
RAL pathway activation, in both wild-type and mutant Ras situations
(Athuluri-Divakar et al., 2016). Inhibition of multiple Ras-mediated
diseases have been seen in response to rigosertib, including pancre-
atic cancer and leukaemia (Athuluri-Divakar et al., 2016; Baker
et al., 2019). Rigosertib is moderately to well tolerated in clinical trials
thus far, although is yet to be specifically tested in the context of
Ras-mutant cancer (Bowles et al., 2014; Ma et al., 2012; Navada
et al., 2020). As with the antibody-derived, small molecule protein–
protein interaction inhibitors described above, toxicity may be as a
result of rigosertib's ability to target both wild-type and mutant KRAS
and thus, further studies are needed to fully assess the impact of such
a drug on healthy cells.
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14. Antibody therapy is also currently being explored as means of
direct Ras targeting. However, a major drawback has been the capa-
bility of the antibody to cross the cell membrane, as with any protein-
based therapy (Bolhassani et al., 2017). Therefore, the development
of inRas37, a pan-Ras-targeting antibody, is a considerable step for-
ward in targeting Ras. Although the cellular uptake remains low
(approximately 4%), in vitro and in vivo work has shown promise in the
potential of inRas37 to inhibit both the MAPK and PI3K–AKT path-
ways, in a dose-dependent manner (Shin et al., 2020). Briefly, this drug
binds to integrins αVβ3 and αVβ5 on the cancer cell surface which
then undergo endocytosis. The antibody “escapes” the endosome as a
result of pH-determined cleavage (the antibody is cleaved from the
integrins better at pH 7, the cytoplasmic pH, compared to pH 6.5, the
pH of early endosomes) (Podinovskaia & Spang, 2018; Putnam, 2012;
Shin et al., 2020). The antibody then co-localises with Ras to block the
effector binding site, in a manner similar to the protein–protein inter-
action inhibitors described above. Mutations introduced into the gen-
eral antibody structure render it specific for mutant Ras binding, with
little activity against Ras wild type, thereby limiting on-target toxicity.
inRas37 has greater effect on cells with a greater dependency on Ras
signalling, which can include some tumour cells (Weinstein, 2002; Yi
et al., 2020). Nevertheless, when tested on large tumour models
(spheroids and cell-derived xenograft mice), efficacy decreased con-
siderably (Shin et al., 2020). This therefore shows that treatment with
this drug may be suitable for patients at earlier stages of Ras-mutated
cancer, before tumour burden is too great and reduces drug efficacy.
Thus, it could be used to treat Ras-initiated relapse as soon as it
occurs. However, its utility in eliminating minor subclones prior to
relapse is limited, given the low cellular uptake and the need for a high
Ras dependency in the cell.
9 | RESISTANCE TO Ras-TARGETING
AGENTS
Of course, there is potential for resistance to any therapeutic and
Ras-targeting drugs are no different. Some of these have been previ-
ously discussed here, such as the use of geranylgeranyltransferase to
overcome the effects of farnesyltransferase inhibitors (Whyte
et al., 1997) or the up-regulation of alternative Ras-mediated path-
ways, as seen when patients are treated with MEK inhibitors (Vitiello
et al., 2019). Other mechanisms of resistance are also possible, such
as the reactivation of ERK, which may be a potential resistance
mechanism in the case of a Ras-targeting agent (Bruner et al., 2017;
Ercan et al., 2012; Ochi et al., 2014). This has already been seen in the
case of EGFR-inhibitor resistance, whereby negative regulators of
ERK are down-regulated, so pro-apoptotic BIM is not fully up-
regulated and so cannot fully induce apoptosis. Alternatively, the gene
encoding ERK1, MAPK1, is amplified. These scenarios resulted in
in vitro and in vivo resistance to the putative EGFR inhibitor WZ4002
(Ercan et al., 2012). ERK reactivation has also been found to contrib-
ute to gefitinib resistance but, in this case, was found to be mediated
by Src (Ochi et al., 2014). Given these kinases are either side of Ras,
their co-operation could result in resistance to a Ras inhibitor. How-
ever, Src-mediated ERK reactivation is avoidable through treatment
with Src inhibitors (Ochi et al., 2014), which may present a means of
overcoming this potential Ras-inhibitor resistance mechanism. Taken
together, these studies imply that whilst resistance to Ras-targeting
drugs is possible, there are already means of overcoming this
resistance, just as a Ras-targeting drug would provide the means of
overcoming Ras-mediated resistance.
Whilst other elements of pathways contributing to resistance can
be targeted relatively easily, acquisition of secondary mutations in Ras
present a more pressing problem. For example, at present, the G12C-
specific inhibitor is perhaps the most developed means of inhibiting
Ras; however, a subsequent mutation in Ras would most likely
render this inhibitor ineffective. This has already been evidenced in
studies into KRAS G12C inhibitors, whereby rescue experiments
with the G12V mutation restored the cancerous phenotype (Ostrem
et al., 2013). Therefore, a pan-mutation-targeting drug, such as a
derivative of inRas37, may be favoured.
10 | DISCUSSION
Ras mutations in cancer and chemoresistance are important when
considering patient prognosis. Whilst it seems inevitable that resis-
tance will be an issue for a long time to come, better targeting of
potential causes is imperative. Advances in Ras inhibition could help
reduce the risk of resistance and relapse for a wide range of cancers,
given its mutational frequency. At present, some success has been
seen when multiple drugs are used to target different pathways impli-
cated in Ras-mutated disease. However, other studies have shown
lack of long-term efficacy when combining multiple therapies. There-
fore, single agent, multi-pathway targeting agents, including direct Ras
inhibitors, are becoming more heavily researched. It will be interesting
to see the effects of these in vivo, since this type of therapy may
reduce the potential for future development of drug resistance by
up-regulation of an alternative pathway. Targeting a common factor
at the centre of multiple pathways may target more cancer cell types
and thus reduce the heterogeneity of the tumour, eliminating
potential resistance causes before they become dominant.
Previous failures of Ras-targeting agents, including farnesyl-
transferase inhibitors, as well as perceived unfavourable protein
dynamics, have resulted in Ras being largely considered undruggable.
However, development of structural analysis techniques and a clearer
understanding of the key residues in Ras has altered this thinking, with
new compounds with novel, allele-specific Ras binding mechanisms
showing great promise. Phase I trials of AMG-510, a first-in-class direct
Ras-targeting agent, suggest a turning point has been reached in this
field of study. Whilst there is much more to be done, these preliminary
data indicate a solution for Ras-mediated resistance may be possible.
Nevertheless, a greater understanding of resistance must be
gained before relapse risk can be eliminated. Despite evidence
supporting the cancer stem cell theory, limited standard-of-care
detection sensitivities for initial diagnostic samples prevent
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15. identification of the minor subclones present at diagnosis (McMahon
et al., 2019). It can therefore be difficult to determine likely causes of
relapse upon initial diagnosis and so constant monitoring for changes
in expression of genes commonly implicated in drug resistance, such
as Ras, may provide a useful tool for predicting disease trajectory.
Nevertheless, this is only useful if the effects of the acquired/emer-
gent mutations can be abrogated. In the cases discussed here,
improved analytical tools would ideally be combined with Ras-
targeting agents to prevent resistance taking hold.
Ultimately, chemoresistance, either intrinsic or acquired, due to
Ras mutations, whether primary or secondary, remains a considerable
problem. The concepts presented here, amongst many other examples,
illustrate the necessity for Ras-targeting drugs. There is a distinct clini-
cal requirement for the improved targeting of Ras in cancer, with Ras
implicated in both initial disease presentation and relapse. Although no
universal, direct Ras inhibitor has yet been achieved, considerable pro-
gress has been made in the last decade with the advent of allele-specific
inhibitors. This brings promise to the field, with the potential for better
treatment of Ras-initiated resistance a real prospect.
10.1 | Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to
corresponding entries in http://www.guidetopharmacology.org and
are permanently archived in the Concise Guide to PHARMACOLOGY
2019/20 (Alexander, Kelly, Mathie, Peters, et al., 2019; Alexander,
Fabbro, Kelly, Marrion, et al., 2019; Alexander, Fabbro, Kelly, Mathie,
et al., 2019).
CONFLICT OF INTEREST
The authors declare no conflicts of interest.
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2860 HEALY ET AL.
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![2 | WHAT IS DRUG RESISTANCE?
There are two general classes of resistance, intrinsic and acquired.
Intrinsic resistance occurs due to overt pre-existing factors, including
variations in protein expression levels (such as increased expression of
the P-gp [MDR1] transporter), epigenetic modifications (including by
the long non-coding RNA (lncRNA) HAND2-AS1 and chromatin modi-
fier Jarid1A (lysine demethylase 5A)) and somatic mutations (such as
in Ras) (Burrell et al., 2013; Gruber et al., 2012; Marusyk et al., 2012;
Sharma et al., 2010). Changes conferring drug resistance often co-
exist, resulting in a resistance heterogeneity similar to that seen in the
original disease itself (Gerlinger et al., 2012; Ramirez et al., 2016).
Acquired resistance is thought to occur for a number of reasons,
including through pre-existing (but initially undetectable) and de novo
mutations (Bhaduri et al., 2020; Russo et al., 2019). This is often
identified weeks to months after treatment has commenced (Santoni-
Rugiu et al., 2019). In recent times, the concept of disease clonal
heterogeneity has provided greater insight into the causes of acquired
resistance, suggesting that chemoresistance and disease relapse occur
as a result of minor subclonal populations. Given that these likely
contribute to the heterogeneous nature of cancer, it seems likely that
these also contribute to disease re-emergence and relapse (Bonnet &
Dick, 1997; Gerlinger et al., 2012; Pattabiraman & Weinberg, 2014;
Roy & Cowden Dahl, 2018; Seth et al., 2019). Within these minor
clonal populations, mutations conferring drug resistance may exist at
the early stages of disease but remain dormant and undetectable upon
first presentation (Pietrantonio et al., 2017; Russo et al., 2018). When
the bulk of the cancer is eliminated as a result of initial chemotherapy
targeted at overt mutations, cells from this minor subclone proliferate
and become dominant in the tumour bulk (Jones et al., 2019;
McMahon et al., 2019).
A second, related, resistance mechanism involves a very rare
subpopulation of cells, known as cancer stem cells. Cancer stem cells
were first described in acute myeloid leukaemia but have since been
applied to many other cancers including (but not limited to) breast
cancer, colorectal cancer, myeloma and pancreatic adenocarcinoma
(Bonnet & Dick, 1997; Koury et al., 2017; Lapidot et al., 1994).
Cancer stem cells have unique properties compared to bulk tumour
cells. They are undifferentiated and have strong self-renewal and
proliferative capabilities and typically remain in a quiescent state,
thus avoiding chemotherapy targeted at rapidly dividing cells (as in
bulk tumour cells). However, they do have the proliferative capacity
to maintain and expand the tumour burden, as bulk tumour cells are
eliminated (Jordan et al., 2006). These “stemness characteristics”
render this subset of cells intrinsically resistant to chemotherapy,
with distinct immunophenotypic and molecular signatures (Bonnet &
Dick, 1997). This includes increased expression of efflux trans-
porter P-gp (MDR1) and the ability to repair damaged DNA, a
common method of inducing cancer cell death (Dean et al., 2005;
Pattabiraman & Weinberg, 2014).
Certain pathways up-regulated in cancer stem cells have been
linked to the increased self-renewal capacity seen in this subset of
cells. Key examples include the Wnt/β-catenin and Hedgehog
(Hh) pathways, as well as the Ras-dependent MAPK and PI3K/AKT
pathways. Of these, the Wnt/β-catenin pathway is perhaps the most
associated with cancer stem cells. Briefly, at a physiological level, Wnt
signalling is not active and β-catenin is ubiquitinated and sent for
proteasomal degradation following interaction with GSK3β, APC and
Axin. In cancer stem cells however, activation of Wnt signalling
inhibits formation of the β-catenin–GSK3β–APC–Axin complex,
stabilising β-catenin. This translocates to the nucleus whereby it
stimulates transcription of proliferative genes, including c-Myc. This
promotes proliferative signalling, increasing the self-renewal capacity
of cancer stem cells (Krausova & Korinek, 2014; Moon et al., 2014).
Although still ambiguous, it has been reported that β-catenin
stabilisation can promote Ras stabilisation and protects Ras against
proteasomal degradation (Jeong et al., 2018; Lee et al., 2018). This in
turn promotes MAPK and PI3K pathway signalling, further contribut-
ing to the self-renewal capacity of cancer stem cells.
Alternatively, up-regulation of the hedgehog (Hh) signalling
pathway has been shown in cancer stem cells. Whilst there are many
facets to this pathway, its activation can stimulate up-regulation of
stemness-associated transcription factors including NANOG, OCT4
and SOX2, thus promoting self-renewal (Boyer et al., 2005; Po
et al., 2010; Takahashi & Yamanaka, 2006; Zhu et al., 2019). Increased
activation and stabilisation of these genes (through either Hh signal-
ling or biochemical stresses) have been implicated in the dedifferentia-
tion of bulk tumour cells to cancer stem cells (Herreros-Villanueva
et al., 2013; Kumar et al., 2012).
Interest in epigenetic remodelling has grown considerably in the
last decade and it has considerable effects in promoting drug resis-
tance. lncRNAs are also implicated in survival of cancer stem cells. In
hepatocellular carcinoma, the lncRNA HAND2-AS1 is up-regulated
and stimulates the self-renewal capacity of cancer stem cells, through
activation of the bone morphogenetic protein (BMP) signalling path-
way. BMP activation has in turn been shown to induce
chemoresistance in other cancer models, such as lung cancer (Gruber
et al., 2012; Wang et al., 2015). Indeed, this pathway is also regulated
by the fusion gene CBFA2T3-GLIS2 and the Hh and JAK-STAT signal-
ling pathways (Gruber et al., 2012; Okada et al., 2014).
Taken together, it is evident that many signalling pathways and
associated factors play a critical role in mediating drug resistance.
Identifying commonalities between these pathways could present an
opportunity to reduce the incidence and impact of drug resistance.
Although the incidence of Ras mutations in cancer stem cells is rela-
tively low, Ras-mediated pathways have been implicated (Corces-
Zimmerman et al., 2014). In addition to the aforementioned β-catenin-
mediated stabilisation of Ras, stabilised OCT4 and SOX2 expression
has also been shown in response to AKT activation, by extracellular
biochemical and radiation stresses (Maiuthed et al., 2018; Park
et al., 2021). Indeed, activation of the MAPK pathway through the
Ras–Raf interaction promotes increased expression of SOX2 and
NANOG (Chan et al., 2018; Du et al., 2019). Furthermore, correlation
has been seen between up-regulation of the MAPK, PI3K and BMP
pathways in drug-resistant lung cancer (Wang et al., 2015). Thus, not
only can these pathways contribute to an increased cellular
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